WO1998007020A1

WO1998007020A1 - Nitric oxide sensor

Info

Publication number: WO1998007020A1
Application number: PCT/US1997/013433
Authority: WO
Inventors: Babs R. Soller
Original assignee: University Of Massachusetts
Priority date: 1996-08-13
Filing date: 1997-07-29
Publication date: 1998-02-19
Also published as: AU3901497A

Abstract

A sensor (10) for sensing nitric oxide includes a chamber (30) attached in fluid communication with a flow line (18), e.g., a ventilation line containing a gas mixture, and an optically transparent element (40) including a sensing compound, e.g., an organometallic compound, that reacts with nitric oxide located within the chamber. The chamber is configured to permit transmission of light through the element. A detector (44) can measure a change in optical character of the optically transparent element and convert the change into a measurement of the presence or concentration of nitric oxide in the gas mixture. The optically transparent element can be in the form of a film, gel, or liquid.

Description

NITRIC OXIDE SENSOR Background of the Invention The invention relates to nitric oxide sensors, and, more particularly, to nitric oxide sensors for measuring nitric oxide in fluids, e.g., gases used in therapeutic treatments for respiratory distress. Nitric oxide (NO) has been reported to have important roles in diverse fields, ranging from neuroscience to urology and cardiovascular medicine, and was named "molecule of the year" in 1992 (Koshland, Science, 258:1861- 1865, 1992). Interest in NO mainly stems from its role in relaxing blood vessels, prompting its therapeutic use as a vasodilator. Currently, NO is being used therapeutically as an inhaled agent because it acts as a selective pulmonary vasodilator and is delivered preferentially to well-ventilated areas of the lung. It is superior to intravenous pulmonary vasodilators which can reduce systemic arterial pressure to dangerous levels in addition to reducing pulmonary resistance. Other inhaled pulmonary vasodilators can worsen arterial hypoxemia because they are delivered to the entire pulmonary vasculature including injured or poorly ventilated areas. For these reasons, nitric oxide has received great interest for the treatment of pulmonary hypertension.

Inhaled nitric oxide has been used successfully to reverse severe persistent pulmonary hypertension in the newborn (PPHN) , a relatively common cause of newborn respiratory compromise. It has recently been demonstrated that the availability of inhaled NO significantly reduces the need for extracorporeal life support in newborns affected by PPHN. Inhaled NO has also been used as rescue therapy for patients suffering from severe adult respiratory distress syndrome (ARDS) or from acute lung injury to reverse pulmonary hypertension. In spite of these benefits, NO also has certain undesirable aspects. Most importantly, the concentration of NO must be carefully measured and controlled in an inhaled gas mixture, because the oxides of NO are toxic. Current technology to measure nitric oxide utilizes chemiluminescence or electrochemical sensors. Chemiluminescence is sensitive to NO concentrations, but requires large, bulky, and expensive equipment. The electrochemical sensors are less expensive; however, these sensors drift over time, require frequent recalibration, and can have long response times for decreasing NO concentrations. Both measurement techniques can underestimate the nitric oxide concentration. Biomolecules that selectively bind to NO have been used to sense NO in solutions. Methods for immobilizing biomolecules include covalent binding, physical adsorption, or cross-linking to a suitable carrier matrix, and physical entrapment and microencapsulation in a polymeric or Si0₂ matrix (sol-gel) (Dave et al., Anal. Chem., 66:1120A-1127A, 1994). The use of hemes and porphyrins immobilized by the sol-gel method have been investigated in the development of NO sensors. For example, Eguchi et al., Sensors and Actuators, B1:154-157 (1990) , describes the investigation of various metal ion- doped porphyrins immobilized onto the end of a fiber for the measurement of nitric oxide from combustion products. Co-doped porphyrin was most sensitive to nitric oxide in the range of 10-1000 mM at 200 'C. Eguchi et al. monitored the change in optical absorption at 420 nm, attributing this change to the oxidation of Co(I) and the formation of Co(II)-N0 complex. Dave et al. (1994), supra. report the use of sol-gel immobilized manganese myoglobin for measurement of NO in solution. Summary of the Invention The present invention provides a reliable, inexpensive means for measuring the concentration of nitric oxide in a fluid, e.g., a gas mixture used for ventilating a subject. A sensing compound is employed which reacts with nitric oxide and undergoes a change in its optical properties upon reacting. Optical spectroscopy methods are used to provide a measure of the nitric oxide concentration. In general, the invention features a sensor for sensing nitric oxide in a fluid. The sensor includes a chamber attached in fluid communication with a flow line, e.g., a gas ventilation line or a liquid ventilation line, containing the fluid, and an optically transparent element located within the chamber. The optically transparent element contains the sensing compound that reacts with nitric oxide. The chamber is configured to permit transmission of light through the element. The change in the optical character of the sensing compound indicates the presence of nitric oxide.

Preferred embodiments include the optically transparent element being in the form of a film, gel, or liquid. A mounting device can be included to position the element within the chamber. A light source can be used to transmit light through the optically transparent element. The sensor can include a light detector which generates an electrical signal in response to light interacting with the optically transparent element, and a processor for converting the electrical signal into a nitric oxide concentration.

The nitric oxide sensing compound can be an organometallic compound containing iron, manganese, chromium, cobalt, platinum, osmium, or ruthenium, as well as oxyhemoglobin, cytochrome c, myoglobins, substituted manganese tetraphenylporphyrins (MnTPP(X)) or chromium tetraphenylporphyrins (CrTPP(X) ) (where X can be Cl, CN, or acetate) , or chlorotetraphenylporphinatoiron(III) (FeCl(TPP)). The compound is preferably immobilized in a polymeric material such as a fluorinated polymeric material permeable to nitric oxide or an organic gel, such as agar.

According to another aspect of the invention, the presence of nitric oxide in a fluid, e.g. , a gas, is detected by placing the sensor in fluid communication with a flow line, delivering light to the sensor, detecting light transmitted through the optically transparent element to generate an electrical signal, and processing the electrical signal to determine the presence of nitric oxide. Preferred embodiments include calibrating the sensor with known concentrations of nitric oxide to generate a calibration equation. The method can include processing the electrical signal with the calibration equation to calculate a concentration of nitric oxide. In a preferred embodiment, the concentration of nitric oxide delivered through a flow line is determined by inducing a change in an optical property of a nitric oxide-sensing compound with nitric oxide in the flow line, and detecting the change in the optical property to determine the amount of nitric oxide. The change in the optical property is detected by delivering light to the nitric oxide-sensing compound, detecting a portion of light that passes through the nitric oxide-sensing compound, and analyzing the collected portion of light to determine the concentration of nitric oxide. The nitric oxide induces a change in the absorption spectrum of the nitric oxide-sensing compound which is related to a concentration of nitric oxide in a fluid delivered to the subject. In another preferred embodiment the presence of nitric oxide in a flow line is determined by inducing a change in an optical property of a nitric oxide-sensing compound with nitric oxide from the flow line, and detecting the change in the optical property to determine the presence of nitric oxide in the flow line.

Advantages of the nitric oxide sensor include low cost, small size, freedom from drift, rapid response, e.g., on the order of 3 to 10 seconds or less, ease of use, and the ability to quickly exchange sensing elements to provide a long-term response.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings Fig. 1 is a diagrammatic representation of a ventilation system including a nitric oxide sensor. Fig. 2 is an illustration of a nitric oxide sensor, according to the invention. Fig. 3 is an absorption spectra of a nitric oxide sensing element.

Fig. 4 is a diagrammatic representation of a test apparatus for testing a nitric oxide sensor. Fig. 5 is an absorption spectra of a non-reacted and a reacted nitric oxide sensing element.

Detailed Description Referring to Fig. 1, a nitric oxide sensor 10 for measuring nitric oxide concentration in inhaled ventilation fluid is connected to a ventilation line 12 leading from a ventilator 14 to lungs 16 of a subject. Nitric oxide flows into sensor 10 through sensor flow inlet line 18 connected to ventilation line 12, and exits sensor 10 through sensor flow outlet line 20 leading to a vent 22 attached to, for example, a standard ventilator scrubber (not shown) . A processor 24 receives a signal from sensor 10 along data line 26 and converts this signal to NO concentration.

Referring to Fig. 2, nitric oxide sensor 10 includes a chamber 30 in fluid communication with flow inlet line 18, and a stack of optically transparent sensing elements 40 located within chamber 30. Alternatively, one sensing element 40 can be used and the shape and form of the sensing element can vary as described further below. Light 41 from a light source 42, e.g., two light emitting diodes, one at a test wavelength and the other at a reference wavelength, discussed further below, enters chamber 30, passes through one or more sensing elements 40 (here, four) , and is detected by a detector 44, e.g., a silicon photodetector, which sends signals to processor 24. Sensing element or elements 40 are located within chamber 30 to intersect the path of the light. The sensing elements can be mounted within the chamber by any suitable device that does not interfere with the transmission of light through the sensing elements, e.g., by a multi-compartment tray 46.

Contained within sensing element 40 is a nitric oxide sensing compound whose optical properties change as a function of NO concentration. A wide variety of known nitric oxide sensing compounds, e.g. , hemoglobin, cytochrome c, manganese myoglobin, or metal containing compounds whose optical properties change permanently or reversibly upon reaction with nitric oxide can be employed in sensing element 40. Many metal-centered hemes and porphyrins, such as plain or substituted porphyrins containing manganese, iron, cobalt, ruthenium, or chromium, for example, manganese tetraphenylporphyrin as well as biologically based hemes, such as the salivary heme protein from β. prolixus. can be used.

Sensing element 40 may take many forms, e.g., a film, a gel, or a liquid. Sensing element 40 can include a polymer matrix in which the sensing compound is embedded. The matrix material is selected to be transparent in the wavelength regions of interest and to be highly permeable to NO. Polymers having the required properties and varying in permeability to NO include Nafion®, polymethyl methacrylate (PMMA) , polyhydroxyethylmethacrylate (PHEMA) , polyvinyl alcohol (PVA) , and siloxane polymers. Nafion®, a tetra fluoroethylene-sulphonyl-fluoride vinyl ether copolymer with the sulphonyl fluoride groups hydrolysed to -SD₂H acidic groups, has been used as a membrane for electrochemical NO sensors and has a high NO permeability. PMMA is also highly permeable to gaseous molecules and forms good films. PHEMA has been used to provide more hydrophilic films and gels with high permeability of gases. PVA has been used with chemical cross-linking as a support for immobilized enzyme systems with high permeability and a high surface area through the formation of thin fibers.

Nitric oxide is highly reactive with heme compounds which have a number of optical transitions that can be adapted for sensing. Thus, heme compounds are preferred NO sensing compounds. Many of these heme compounds include Fe(II), which is readily oxidized to Fe(III) by NO and can be monitored by absorption spectroscopy. Additionally, Fe(III) forms a stable complex with NO, Fe(III)-N0, resulting in the appearance of a new absorption band.

Nitric oxide concentrations can be calculated by measuring the light transmitting properties of a heme compound at any wavelength where the nitric oxide reaction product absorbs (417 nm, 526 nm, and 564 nm for the cytochrome c-NO complex) . The reference LED, being at a wavelength where the reaction product does not absorb, provides a reference signal to monitor the incident light intensity. With the heme compound incorporated into sensing element 40, the amount of light transmitted through sensing element 40 is measured and converted to NO concentration using mathematically derived calibration curves.

The following is a procedure for forming sensing element 40 as a film, for example, of cytochrome c immobilized in polyvinyl alcohol: 1) a solvent is prepared by adding EtOH to ddH20 (50% v/v) and stirring until well mixed; 2) a heme compound is prepared by adding horse-heart cytochrome-c to the solvent and stirring until the heme is dissolved; 3) the film is prepared by putting the heme solution in a large beaker, and, while stirring, slowly adding PVA to the heme solution until the PVA is well dissolved and there are no clumps; 4) the solution is then poured into an appropriate drying container, e.g., a glass cuvette; and 5) the solution is dried at 40°C in a non-convection oven for about 3 to 5 days to slowly evaporate the solution. Referring to Fig. 3, an absorption spectrum of a film produced as described above is shown. The absorption spectrum is unchanged from an absorption spectrum of a solution of the same material.

To form a gel, agar is heated to boiling, cytochrome C is mixed in when the agar cools to 40°C, and the solution then gels as it cools further. Films of varying thickness can be made by controlling the volume of the agar applied to a smooth surface. To use the sensor in liquid form, steps 1 through 3 described above are followed. The liquid sensor can be held in a gas permeable and optically transparent container with the ventilation gas bubbled into the solution. To increase the surface area of sensing element 40, when formed as a film or gel, the sensing element may be drawn into fibers and bundled, used in a stack, as shown in Fig. 2, or coated onto an optically transparent mesh. Sensor 10 provides a measurement of the change in concentration of nitric oxide within the ventilation fluid with each breath over time. During inhalation of nitric oxide, sensor 10 can be used as a safety monitor by measuring levels of inhaled nitric oxide to prevent reaching toxic levels and as a monitor for ventilator or ventilation delivery device failure by signalling when no nitric oxide is being delivered. The sensor also provides a quick response, on the order of 3 to 10 seconds or less, to sudden increases and decreases in nitric oxide levels within the gas mixture.

Referring to Fig. 4, an experimental apparatus 110 for testing sensing element 40 includes air, oxygen, nitrogen, and nitric oxide sources 160, 162, 164, and 166, respectively, with associated pressure/flow regulators 168. An in-line flow meter 170 measures the concentration of the air and oxygen mixture delivered by gas mixer 172 to gas mixer 174, a second in-line flow meter 170a measures the concentration of the nitrogen delivered to gas mixer 174, and a third in-line flow meter 170b measures the concentration of the nitric oxide delivered to gas mixer 174. The resulting gas mixture enters a sensing chamber 130 through inlet line 118 via inlet port 119, and exits the chamber via outlet port 121 to outlet line 120 leading to vent 122. Light of the appropriate wavelength is delivered via a fiber optic cable 150 through a lens 151 to chamber 130, and transmitted light is received through a lens 152 to a second fiber optic cable 153 in communication with a spectrophotometer 154. Sensing element 40 is located within chamber 130 to intersect the path of light from fiber optic cable 150 to fiber optic cable 153. The sensing element may be mounted by any device that does not interrupt the path of the light through the sensing element, e.g., by clips 146. The experimental apparatus can be used to calibrate sensing element 40, i.e., to determine the equation that relates NO concentration to the change in optical properties of the sensing element. The optical absorption (A) is measured by the monitor (A = log(l₀/I), where I₀ is the incident light intensity impinging on the sensing element and I is the light intensity transmitted through the sensing element) . The optical absorption is related to NO concentration by the Beer-Lambert law:

elc

where e is the molar extinction coefficient for the sensing compound, 1 is the effective pathlength of the light as it travels through the sensing element, and c is the concentration of NO. To derive a calibration equation for each sensing element the element is placed in the test chamber and A is measured for at least 3 known concentrations of nitric oxide. Using linear regression, the slope is determined with A as the y variable and c as the x variable. Once the slope (equal to el ) is known, the NO concentration of an unknown gas mixture can be determined.

Example A film is formed by dissolving 1 gram of agar in 100 ml of deionized water. The solution is heated until it boils and dissolves the agar. When the temperature of the solution drops to 37°C, 100 ml of the solution is removed and 125 mg of cytochrome c is dissolved in the solution. 20 ml of the cytochrome c solution is placed in a polystyrene tray (5" x 3.5") and dried for approximately 2 days to produce a sheet approximately 100 μm thick. This sheet is cut into film pieces approximately \* x ¥ square and twelve of the film pieces are stacked approximately 2 mm apart. To test the films, referring to Fig. 5, the spectra 202 of the unreacted film is first recorded. The films are then reacted with a continuous flow of nitric oxide (1000 ppm in nitrogen) . As seen in Fig. 5, the single absorption peak 204 at 530 nm in the spectra 202 of the unreacted cytochrome c, after reaction with NO and normalization of the resulting spectra by removing the component of the spectra associated with unreacted cytochrome c, splits into two absorption peaks 206 and 208 at 526 nm and 564 nm, respectively, in the spectra 210 of the reacted cytochrome c. The change in the absorption spectrum of the cytochrome c after reaction with NO, as represented by absorption spectra 210, enables the concentration of NO to be measured. To use sensor 10, light with a wavelength at one of the absorption peaks of the reacted sensing compound is directed onto the film and the transmitted light is detected. The peak wavelength is selected to provide the greatest sensitivity to change in the absorption of the cytochrome c upon reacting with NO. The resulting spectra is normalized by removing the component of the spectra associated with unreacted cytochrome c. The remaining signal is a measure of the concentration of NO. Sensing element 40 can include a nitric oxide sensing compound that irreversibly or reversibly reacts with nitric oxide. Monitoring of nitric oxide concentration over a long period of time with a compound that irreversibly reacts with nitric oxide can be accomplished by using a high concentration of the compound, by using a sensing element that is easily replaced, or by using a sensing element in the form of a roll of film that can be fed through the sensing chamber. Fiber optics can be used to deliver light to sensing element 40 and receive the transmitted light.

Such a system is described in co-pending application U.S. Serial No. 08/347,875, filed December 1, 1994, hereby incorporated by reference.

Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

For example, referring to Fig. 1, ventilator 14 can be a liquid ventilator with nitric oxide sensor 10 measuring the nitric oxide concentration in the ventilating liquid, e.g., a perfluorocarbon.

Claims

What is claimed is:

1. A sensor for sensing nitric oxide in a gas, the sensor comprising: a chamber adapted to be attached in communication with a flow line containing the gas, and an optically transparent element contained within said chamber, said element including a sensing compound that reacts with nitric oxide, an optical character of said sensing compound changing upon reaction with nitric oxide, said chamber being configured to permit transmission of light through said element, wherein the change in the optical character of said sensing compound indicates the presence of nitric oxide in the gas.

2. A sensor of claim 1, wherein said flow line comprises a ventilation line delivering ventilation gas to a subject.

3. A sensor of claim 1, wherein said element comprises a film, gel, or liquid.

4. A sensor of claim 1, further including a mounting device for positioning said element within said chamber.

5. A sensor of claim 1, further comprising a light source for transmitting light through said optically transparent element.

6. A sensor of claim 5, further comprising a light detector for generating an electrical signal in response to light interacting with said optically transparent element.

7. A sensor of claim 6, further comprising a processor for converting the electrical signal to a nitric oxide concentration.

8. A sensor of claim 1, wherein said sensing compound is an organometallic compound.

9. A sensor of claim 8, wherein said sensing compound comprises iron, manganese, chromium, cobalt, platinum, osmium, or ruthenium.

10. A sensor of claim 1, wherein said sensing compound is oxyhemoglobin.

11. A sensor of claim 1, wherein said sensing compound is cytochrome c.

12. A sensor of claim 1, wherein said sensing compound comprises a myoglobin.

13. A sensor of claim 1, wherein said sensing compound is selected from the group consisting of a manganese tetraphenylporphyrin and a chromium tetraphenylporphyrin.

14. A sensor of claim 1, wherein said sensing compound is chlorotetraphenylporphinatoiron(III) .

15. A sensor of claim 1, wherein said optically transparent element comprises a polymeric material in which said sensing compound is immobilized.

16. A sensor of claim 15, wherein said polymeric material comprises a fluorinated polymeric material permeable to nitric oxide.

17. A sensor of claim 15, wherein said polymeric material comprises an organic gel.

18. A method of detecting the presence of nitric oxide in a gas in a flow line, the method comprising placing the sensor of claim 1 in fluid communication with the flow line, delivering light to the sensor, detecting light transmitted through the optically transparent element to generate an electrical signal, and processing the electrical signal to determine the presence of nitric oxide in the gas.

19. A method of claim 18, further comprising calibrating the sensor with known concentrations of nitric oxide to generate a calibration equation.

20. A method of claim 19, wherein said step of processing includes processing the electrical signal with the calibration equation to calculate a concentration of nitric oxide.

21. A method of claim 18, wherein said step of placing the sensor includes placing the sensor in fluid communication with a gas ventilation line.

22. A method for determining the presence of nitric oxide in a gas, the method comprising: inducing a change in an optical property of a nitric oxide-sensing compound with nitric oxide in the gas; and detecting the change in the optical property to indicate the presence of nitric oxide in the gas.

23. A method of claim 22, wherein said detecting step comprises delivering light to the nitric oxide- sensing compound, detecting a portion of light that passes through the nitric oxide-sensing compound, and analyzing the collected portion of light to determine the concentration of nitric oxide in the gas.

24. A method of claim 22, wherein the nitric oxide induces a change in the absorption spectrum of the nitric oxide-sensing compound.

25. A method of claim 24, wherein a change in the absorption spectrum is related to the concentration of nitric oxide in the gas.

26. A method of claim 22, wherein said inducing step includes inducing a change in the optical property of the nitric oxide-sensing compound with nitric oxide in a gas ventilation line.